This application relates to inducible transcription control sequences (TCS). In particular, it relates to inducible TCS for metallothionein (hereinafter "MT"), specifically yeast MT.
With the advent of recombinant DNA research, the need has arisen for strong and efficient promoters to direct the synthesis of proteins in microbial culture.
Yeast organisms are well suited for commercial expression of heterologous proteins, having advantages over tissue culture and bacteria. First of all, highly developed technology exists for yeast fermentation processes. Yeast can be employed safely since handling and disposal methods are well established. In addition, yeast is an eukaryotic organism which has advantages when expressing genes originating from other eukaryotic backgrounds. Most of all, yeast is extremely well characterized and can be manipulated genetically to maximize expression of heterologous peptides.
The ideal expression system for yeast as a microbial "factory" would allow for enhanced protein production, controlled during portions of the fermentation process. Products are currently made utilizing multi-copy plasmids, efficient promoters and amplified genes. However, these systems still lack the ideal parameter, which involves the ability to direct product synthesis at a specific time during the fermentation. By divorcing the growth process (which may involve days of culture incubation) from that of product synthesis (which occurs within hours), one can obtain higher levels of product. The product is exposed to less time in the media or within cells where it may be susceptible to proteolytic degradation, and any detrimental effect of the product on the growth of the organism is removed by largely separating the growth and production processes. Controllable promoters will lead to efficient cell recycling and product harvesting during continuous culturing of cells.
We have found that MT TCS (specifically copper chelatin from yeast) are useful for this type of application. Metallothionein is known to be rapidly induced by the addition of copper ions to the media. Within ten minutes of exposure to copper the promoter is functioning maximally and the culture is synthesizing MT. The copper inducibility is retained on autonomously replicating plasmids in yeast.
For the purposes herein, TCS is defined as the combination of a promoter with a metal ion inducing region.
Publications which should be consulted in regard to the disclosures herein are Fogel et al., 1982, "P.N.A.S.(USA)" 79:5342-5346; Carter et al., 1983, in Current Communications in Molecular Biology (eds. Gluzman, Y. and Shenk, T.) p. 170 (Cold Spring Harbor, N.Y.); Valenzuela et al., 1983, in The Molecular Biology of Yeast (eds. Hicks, J. B., Klar, A. and Strathern, J. N.) p. 255 (Cold Spring Harbor, N.Y.); Butt, 1983, "J. Cell Biochem. Supp." 7A: 384; Butt et al., 1984, "Gene" 27:23-33; Anonymous, in Genetic Engineering News 3(2) p. 6 (1983); Baxter et al., European Pat. Application 78,154; Fogel et al., European Patent Application 96,491; Welch et al., 1983 in The Molecular Biology of Yeast (eds. Hicks, J. B., Klar, A. and Strathern, J. N.) p. 249 (Cold Spring Harbor, N.Y.), p. 249; Welch et al., 1983, "Mol. Cell Biol." 3 (8): 1353-1361; and Karin et al., 1984 "P.N.A.S. (USA)" 81: 337-341.